KR940009281B1 - Alpha-hanp recognizing monoclonal antibody and immunologically measuring reagent for alpha-hanp - Google Patents

Abstract

This invention relates to a class of monoclonal antibody which recognizes an epitope in the N-terminal half of the ring structure of alpha -hANP, to hybridomas producing such antibodies, and to their use in immunoassay and kits containing them.

Description

Monoclonal antibodies that recognize α-hANP and reagents for immunoassay of α-hANP

1 shows cross-reactivity of α-hANP, α-rANP and α-hANP fragments with monoclonal antibodies of the invention.

2 shows the standard curve of α-hANP (○) and the dilution curve of plasma (●, ▲, ■) by EIA.

3 shows the standard curve of α-hANP (○) and the dilution curve of plasma (●, ▲, ■) by one step EIA.

The present invention relates to monoclonal antibodies recognizing α-hANP and hybridomas producing such monoclonal antibodies. In particular, the present invention relates to monoclonal antibodies which recognize near the N-terminal half of the ring structure of α-hANP containing Met [12], and hybridomas producing such monoclonal antibodies. The present invention also relates to an α-containing antibody immobilized on a solid phase recognizing the N-terminal side or the C-terminal side of α-hANP and an enzyme-labeled antibody recognizing a side different from the side recognized by the fixed antibody. It relates to a reagent for enzymatic immunoassay of hANP (hereinafter abbreviated as EIA).

Atrial sodium diuretic polypeptide (ANP) is present in granules produced by muscle cells of atrium cordis and has potent diuretic and sodium diuretic action. This type of polypeptide is found in mice as well as humans, called hANP and rANP, respectively. Each hANP and rANP is further classified into three types-α, β and γ. It consists of the 28 amino acid residues of a-hANP. Cys [7] and Cys [23] at the 7th and 23rd positions from the N-terminus, respectively, form disulfide bonds, and the sequences there between form a ring structure [Biochem. Biophys. Res. Commun. (Hereinafter abbreviated as BBRC) 118, 131-139. 1984].

α-rANP is different from α-hANP and only one residue at position 12 from the N-terminus is Ile in α-rANP and Met in α-hANP (BBRC 117, 839-1983). β-hANP is an antiparallel dimer of α-hANP (Japanese Patent Laid-Open No. 184098/1985). γ-hANP consists of 126 amino acid residues, wherein the C-terminal 99-126 amino acids correspond to α-hANP. As a measuring method of ANP, radioimmunoassay using antiserum is already known (Science 228, 323-325, 1985; Nature 314, 264-266, 1985; BBRC 124, 815-821, 1984; BBRC 124, 663-668, 1984; BBRC 125, 315-323, 1984). Of these, antiserum CR-3 is known to recognize the C-terminal fragment [17-28] of ANP.

As a monoclonal antibody which recognizes (alpha) -hANP, 11A-A11 is known. The antibody is obtained using atriopeptin II (a kind of murine ANP) as an antigen, whose epitope is located between Cys @ 7] and Ser [25] and contains disulfide bonds. Therefore, epitopes are believed to be part of the ANP ring structure. However, the affinity of the antibody is not related to the difference between Met [12, human] and Ile [12, rat], and the antibody recognizes rANP and hANP (Life Science 38, 1991-1997, 1986).

In conventional immunoassays of ANP, ANP must be extracted from plasma samples. Therefore, there is a strong demand for monoclonal antibodies with high affinity such that high sensitivity analysis is possible without extraction. Examples of monoclonal antibodies that recognize ANP include 11A-A11 described above. However, this antibody recognizes not only hANP but also rANP. On the other hand, as described above, antiserum CR-3 against ANP recognizes the C-terminal side of ANP. Therefore, if an antibody that specifically recognizes the N-terminal side of ANP can be obtained, a sandwich immunoassay using these two types of antibodies can be performed. For this reason, there is a strong demand for monoclonal antibodies that specifically recognize the N-terminal side of hANP and have high affinity.

As immunoassays for hANPs, radioimmunoassays (hereinafter abbreviated as RIA) using radioisotopes and enzymatic immunoassays (EA) using enzymes have been known. In RIA, there was a tendency to avoid it because of the necessary equipment and facilities. Therefore, the development of high sensitivity EIA that can be used anywhere is required. As an enzyme-labeling method, glutaraldehyde has generally been used as a crosslinking agent so far. However, these days, it is rarely used due to problems of polymerization and low yield. Reagents such as N-hydroxysuccinimidoesters of N, N'-O-phenylenedimaleimide and N- (4-carboxycyclohexylmethyl) maleimide have recently been known as crosslinking agents instead of glutaraldehyde [J . Immunoassay 4, 209-327 (1983). However, it is not reported that it can be applied to the analysis of hANP. Therefore, it is hard to say that a high sensitivity immunoassay for hANP has been developed.

The present invention relates to hybridomas producing monoclonal antibodies and monoclonal antibodies that recognize the N-terminal half of the ring structure of α-hANP containing Met [12]. In addition, the present invention is an α- containing an antibody immobilized on a solid phase that recognizes the N-terminal side or the C-terminal side of α-hANP and an enzyme-labeled antibody that recognizes a side different from the side recognized by the fixed antibody. It relates to a reagent for the enzyme immunoassay of hANP.

When measuring α-hANP by the conventional RIA method, α-hANP should be extracted from the plasma sample. However, using the reagent for immunoassay of α-hANP using the monoclonal antibody of the present invention, α-hANP can be directly measured in the test sample without extracting α-hANP.

In addition, the antibody immobilized on the solid phase may be measured by a two-step method of first reacting the α-hANP and then further reacting the labeled antibody as well as the one-step method of simultaneously reacting the α-hANP and the labeled antibody with the antibody immobilized on the solid phase. . Therefore, α-hANP can be easily measured. With this measurement of α-hANP, it is easy to accurately diagnose and manufacture heart disease, kidney disease, hypertension (intrinsic or secondary), edema (cirrhosis, nephropathy, sudden seizures, etc.) and dehydration associated with imbalance of body fluids. Can be.

The inventors have worked hard to develop high sensitivity monoclonal antibodies that specifically recognize the N-terminal side of hANP. As a result, a monoclonal antibody that recognizes the N-terminal side of the ring structure of α-hANP was obtained. Furthermore, using this antibody, the high sensitivity analysis of (alpha)-was established.

(1) Preparation of hybridomas producing monoclonal antibodies

It is a polypeptide consisting of 28 amino acid residues of α-hANP and has a relatively low molecular weight. As a result, the ability to induce antibody-production (immunogenic) is low. For this reason, it is combined with bovine serum albumin or bovine thyroglobulin for use as an antigen. The conjugate obtained is emulsified with a suitable adjuvant such as Freund's complete adjuvant and then used to immunize the mouse.

The fluids are immunized with mice inoculated multiple times intraperitoneally, subcutaneously or intravenously every few weeks. Three to five days after the last immunization, the spleen is taken out and used as antibody-producing cells. At the same time, the hybrid antibody to create a hybridoma - a suitable marker such as a cell to be fused with the producing cells, hypoxanthine-guanine-phospholipid view transferase-deficient (HGPRT ^-) or thymidine ^{kinase-deficient} (TK) After removing the myeloma cell line having, the myeloma cell line is fused with antibody-producing cells to prepare hybridomas.

As culture medium for preparing hybridomas, there are mediums such as Eagle MEM, Dulbecco's modified medium and RPMI-1640, which are generally used by adding about 15% fetal bovine serum (FCS) as needed.

First, myeloma and splenocytes are prepared at a ratio of 1: 6.5 as parent cells. As the fusing agent, 50% polyethylene glycol (PEG) is generally used because of the high degree of fusing. Fusion stocks are selected by the HAT selection method. The produced hybridomas are screened by known methods such as membrane fluorescent antibody technique using enzyme culture supernatant, enzyme-linked immunosorbent assay (ELISA) or immunohistostaining. Thus, hybridomaclones that produce the desired immunoglobulin are selected. To prepare hybridomas as monoclonal cells, normal splenocytes are placed in 96-well microwells at 10 ⁵ cells / well in the feed layer, allowing hybridomas to 1 cell / well and screening for regrowing clones. This subcloning is repeated to obtain monoclonal hybridomas.

(2) Preparation of Monoclonal Antibodies

To prepare the monoclonal antibodies of the invention, the hybridomas obtained above are cultured in vitro and in vivo. When culturing in vitro, FCS can be added to the above-mentioned normal medium and used. After incubation for 3 to 5 days in the medium, monoclonal antibodies are obtained in the culture supernatant. For in vivo culture, hybridomas are inoculated into the abdominal cavity of mammals. After 7-14 days, ascites are collected to obtain monoclonal antibodies. Compared to in vitro culture, much larger amounts of antibodies are produced effectively in in vivo culture. Therefore, in vivo culture is preferred.

Monoclonal antibodies obtained from the culture supernatant or ascites are purified by appropriate combination of known methods such as ammonium sulfate fractionation, DEAE Sepharose column and the like. For example, purification is carried out by the method mentioned in the Examples.

As shown in the examples below, the monoclonal antibody KY-ANP-I obtained in the present invention specifically recognizes α-hANP and shows little affinity for rANP. The epitope is believed to be part of the N-terminal half of the ring structure of α-hANP, particularly from Cys [7] to Gly [16]. In addition, since it reacts very weakly with rANP, the epitope is presumed to be the part containing Met [12].

Since the epitope is a part common to β-hANP and γ-hANP as well as α-hANP, the antibody also shows reactivity with β-hANP and γ-hANP.

Moreover, this antibody shows high affinity with (alpha) -hANP (Ka = 3.1 * 10 <^10> M <^-1> ). As shown in the standard curve of α-hANP in FIG. 1, the 10% and 50% inhibitory concentrations (IC ₁₀ and IC ₅₀ ) are ₁₀ pg / vi and 100 pg / vi, respectively.

The hybridoma KY-ANP-I, which produces the monoclonal antibody KY-ANP-I of the present invention, is a PHLS Center for Applied Microbiology & Research, European Collection located in SP4 OJG, Salisbury, Photon, UK under the Budapest Treaty. It has been deposited with accession no. 87082001 to the PHLS Center for Applied Microbiology & Research, European Collection of Animal Cell Culture (ECACC) from August 20, 1987.

(3) binding of the antibody to the carrier

As the solid phase for binding the antibody to the carrier, a carrier such as glass or plastic beads, balls, tubes or plates which are commercially available and generally used in immunoassays as carriers for antigen antibody reaction can be used. Antibodies that recognize the N-terminal side or C-terminal side of α-hANP are adsorbed to either of these carriers. It is usually adsorbed overnight at room temperature in phosphate buffer at pH 6-10, preferably near neutral. The carrier to which the antibody is adsorbed is cold preserved in the presence of a preservative such as sodium azide.

Both monoclonal and polyclonal antibodies can be adsorbed to the carrier in the same manner as above.

(4) rabbit anti α-hANP [17-28] serum

Rabbits were immunized several times with α-hANP [17-28] -sotyroglobulin conjugates prepared by the carbodiimide method, and at 10-14 days after the last immunization, blood was collected and rabbit anti-α-hANP [17- 28] Prepare serum.

(5) Preparation of enzyme-labeled antibody

[Rabbit IgG, F (ab ′) ₂ and Fab ′]

The antiserum prepared as above was fractionated with sodium sulfate and then passed through a DEAE-cellulose column to obtain IgG. The obtained IgG was digested with pepsin to prepare F (ab ') ₂ , followed by reduction with 2-mercaptoethylamine to give anti-α-hANP [17-28] Fab'. Methods for preparing Fab ′ from IgG are described in J. Immunoassay 4, 209-327 (1983), which may be used in the present invention.

[Enzyme-labeling of Antibodies]

As the enzyme to label the antibody, alkaline phosphatase, β-D-galactosidase, peroxidase, glucose oxidase and the like can be used. In the present invention, wasabi peroxidase is preferably used. N, N'-O-phenylene dimaleimide, N-succinimidyl 4- (N-maleimidomethyl) cyclohexanoate, N-succinimidyl 6-maleimidohexanoate, N'- as a crosslinking agent Succinimidyl 3- (2-pyridyldithio) propionate, 4,4'-dithiodipyridine and others are already known. Reaction of this crosslinking agent with an enzyme and an antibody can be performed by a well-known method according to the characteristic of each reagent. Depending on the conditions, antibody fragments such as Fab ', Fab and F (ab') ₂ are used as antibodies. In the present invention, it is preferable to use N-succinimidyl 4- (N-maleimidomethyl) cyclohexanoate or N-succinimidyl 6-maleimidohexanoate. It is also possible to obtain enzyme-labeled antibodies in the same way, regardless of whether they are polyclonal or monoclonal antibodies. Therefore, the enzyme-labeled antibody obtained using the two crosslinkers described above can be represented by the following general formula (I):

[Wherein A represents an antibody or fragment thereof that recognizes the N-terminal side or C-terminal side of α-hANP, B represents a labeling enzyme, and R represents 4-methylene cyclohexyl or pentamethylene.]

The enzyme-labeled antibody thus obtained is preferably purified by affinity chromatography to obtain a high sensitivity immunoassay. Purified enzyme-labeled antibody is stored in a cool dark place with a stabilizer such as thimerosal or glycerol or after freeze drying.

When measuring α-hANP using the immunoassay reagent prepared as above, the antibody is combined as follows: When the antibody which recognizes the N-terminal side of α-hANP is fixed, the C-terminal side is recognized. The antibody is enzyme-labeled. When immobilizing antibodies, relatively large amounts of antibodies are generally required. Therefore, for immobilization, monoclonal antibodies which can be obtained stably in large quantities are suitable (for example, KY-ANP-I of the present invention). However, it is also convenient to use polyclonal antibodies prepared in antisera. As the enzyme-labeled antibody, a monoclonal antibody or a polyclonal antibody can be used as long as it recognizes a site different from that recognized by the immobilized antibody. For example, when KY-ANP-I of the present invention is used as a fixed antibody, the antisera CR-3 described above or the antisera F36 mentioned in the examples can be used as an enzyme-labeled antibody. This combination can also be used inversely. Of course, monoclonal antibodies that recognize the C-terminal side of α-hANP and antisera that recognize the N-terminal side of α-hANP can be used in the present invention.

EXAMPLE

[Production of hybridomas]

Synthetic α-hANP (1.5 mg) and bovine thyroglobulin (5.4 mg) are dissolved in 2 ml of distilled water. 30 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide solution dissolved in 1 ml of distilled water was added dropwise at room temperature over 10 minutes, and then the mixture was stirred at room temperature for 24 hours. . This solution is dialyzed six times for three days in 3 liters of distilled water. The dialysate is divided into 5 fractions and stored at -20 ° C (BBRC 124, 815-821, 1984).

Distilled water was added to the solution stored in the divided fraction (containing 300 μg α-hANP) to make 1.2 ml, and then suspended in 1.2 ml Freund's complete auxiliary solution. Of this, about 2 ml are injected intraperitoneally and subcutaneously into 10BAl / c female mice (200 μl per mouse). Three weeks later mice are further immunized in the same manner. After 5 weeks of additional immunization, antibodies in the blood are measured and two mice with the best immune response are further immunized with the solution containing 50 μg of α-hANP through the tail vein. After 4 days, splenocytes from two mice are collected for cell fusion.

The collected splenocytes (1.3 × 10 ⁷ cells) and myeloma cells × 63 −Ag 8.653 (2 × 10 ⁷ cells) are mixed in Dulbecco's medium (DMEM) and the mixture centrifuged at 1500 rpm, 4 ° C. for 5 minutes. do. The pellet obtained is warmed to 37 ° C. and dissolved, and then 1 ml of 50% PEG 4000 (1 g of PEG / DMEM 1 ml) is added dropwise for 1 minute. After the mixture was left at 37 ° C. for 2 minutes, 10 ml of DMEM was added dropwise at 37 ° C. for 5 minutes to dilute the solution. Centrifuge in DMEM containing 15% FCS at 4 ° C.

The obtained cells are placed in 96-well plates, incubated for 2 weeks in HAT medium, and further cultured for 1 week in HT medium. Hybridomas were propagated in about 30% (212/768) of the total 768 wells, of which 4% (8/212) were found to produce α-hANP antibodies.

Cells in wells showing the highest antibody titers are cloned by limiting dilution. That is, as feed cells, BALB / c mouse thymic cells were added to the wells at 10 ⁶ cells / well and cultured with hybridomas contained at 1 cell / well. This method is performed twice.

With this cloning, the clones that stably produce the highest amount of antibody are selected and named KY-ANP-I.

Antibody titers in supernatants in antisera and hybridoma cultures obtained from immune mice are measured as follows: Supernatants in antisera or hybridoma cultures of immune mice are taken as samples. A mixture of 100 μl dilutions of the sample, 300 μl assay buffer (RIA buffer) and 100 μl of ¹²⁵ I-α-hANP (10000 cpm) was continued to react at 4 ° C. for 24 hours, and then the solution was depleted in 1 ml of Dex. It is mixed with tran-coated-tan and the mixture is reacted at 4 ° C. for 5 minutes. The mixture is centrifuged at 3000 rpm at 4 ° C. for 30 minutes and the radioactivity of the supernatant is measured by a γ-counter tube to calculate the antibody titer in the dilute sample solution.

The ^125- α-hANP described above is prepared by the chloramine T method. That is, α-hANP (1 μg) was mixed with Na ¹²⁵ I (1 mCi), 10 μl of chloramine T (5.25 mg / mL) was added, and after 10 seconds, 20 μl of sodium pyrosulphite (4.5 mg / mL) was added. do. To this was further added 1 ml of 2% gelatin, which was purified by Sep-Pak C 18 from Waters.

[Production of Monoclonal Antibody]

BALB / c mice are pretreated twice at 1-2 week intervals by intraperitoneal injection of 0.5 ml of pristane per mouse at a time. Each mouse is injected intraperitoneally with 5 × 10 ⁶ cells of hybridoma KY-ANP-I suspended in 200 μl of DMEM. The obtained ascites was purified by Protein A-Sepharose CL-AB column to obtain monoclonal antibody KY-ANP-I.

[Characteristics of KY-ANP-I]

The isotype of this monoclonal antibody is determined to be IgG ₁ by the Oucrolone method. A scatter plot based on radial binding analysis was made to obtain the affinity, with the result Ka = 3.1 × 10 ¹⁰ M ⁻¹ .

The epitope is determined by measuring cross-reactivity with various ANP-related peptides by RIA, and the results are shown in FIG. Since it hardly reacts with α-ANP [17-28] and α-ANP [1-6], it is assumed that epitopes are included in α-ANP [7-16]. On the other hand, it reacts very weakly with α-hANP [8-22] or α-rANP. Therefore, it is assumed that the epitope must have a ring structure and the epitope will contain Met. It can be concluded that the epitope recognized by the monoclonal antibody of the present invention is almost N-terminal half of the ring structure of α-hANP.

On the other hand, the reactivity of KY-ANP-I with α-hANP and α-hANP is investigated by the antibody titer measuring method described above. As a result, it was found that KY-ANP-I recognizes not only α-hANP but also α-hANP and also reacts with β-hANP in 80% cross reactivity.

[Preparation of anti α-hANP [17-28] bovine tyroglobulin serum

α-hANP [17-28] is bound to tyroglobulin by the known carbodiimide method [BBRC 117, 695, 1984). To 0.5 ml aqueous solution containing 3.1 mg α-hANP [17-28] was added 0.7 ml aqueous solution containing 19 mg bovine tyroglobulin. To this was added 1 ml of an aqueous solution containing 40 mg of 1-ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride and the mixture was stirred under ice cooling for 2 hours. The reaction solution was dialyzed three times at 4 ° C. with 2 L of distilled water, and then lyophilized to obtain 21 mg of the conjugate. The binding molar ratio of α-hANP [17-28] to bovine tyroglobulin was obtained by amino acid analysis, 30.

[Immunization and Blood Collection]

0.25 mg of the α-hANP [17-28] -sotyroglobulin conjugate was dissolved in 0.25 ml of physiological saline. It is suspended in the same amount of Freund's complete supplement and the solution is injected subcutaneously into at least 20 sites of the rabbit. This method is performed six times at three week intervals, and 10 days after the last injection, rabbits are bled in the carotid artery to obtain antiserum F36.

[Production of Rabbit IgG]

0.18 g of sodium sulfate is slowly added to 1 ml of anti α-hANP (F36) with stirring, and the additive is completely dissolved and stirring is continued at 22 to 25 ° C. The mixture is centrifuged at 10000 rpm at 22-25 ° C. for 10 minutes. The precipitate is dissolved in 1 ml sodium phosphate buffer (pH6.3; 17.5 mmol / l) and then dialyzed with the same buffer. The supernatant is passed through a DEAE cellulose column equilibrated with the same buffer (pH6.3; 17.5 mmol / L). The wet volume of DEAE cellulose required to pass 10 mg of protein in the supernatant is 1 ml. The amount of IgG obtained, calculated using an extinction coefficient of 1.5 g ⁻¹ L cm ⁻¹ and an molecular weight of 150000 of IgG at 280 nm, is 7 mg (J. Immunoassay 4, 209-327 (1983).

[Production of F (ab ′) ₂ ]

10 mg rabbit IgG is dialyzed at 5 ° C. with 1 ml sodium acetate buffer (pH 4.5, 0.1 mmol / l). To the dialyzed IgG solution is added 0.05 mol (1/20 volume) of aqueous sodium chloride solution (2 mol / l). Pepsin (0.2 mg / 10 mg IgG) derived from pig gastric mucosa was dissolved therein. The mixture is reacted at 37 ° C. for 15 to 24 hours. The mixture was then adjusted to pH 8 using aqueous sodium hydroxide solution (1 mol / l), and the mixture was then separated into a Sephadex G-150 column (1.5 × 45 cm for 1.0-1.5 ml, 2.0 × 45 for 2.0-2.5 ml). Cm) and eluted with sodium borate buffer (pH 8.0, 0.1 mol / L). The amount of F (ab ') ₂ obtained, calculated using an extinction coefficient of 1.48 g ^-1 lcm ^-1 and a molecular weight of 92000 of F (ab') ₂ at 280 nm, is 6 mg (J. Immunoassay, 4, 209). 327 (1983).

[Manufacture of Fab ′]

3 mg of F (ab ') ₂ is dissolved in 0.45 ml of sodium phosphate buffer (pH 6.0, 0.1 mmol / l). To this was added 2-mercaptoethylamine / sodium phosphate buffer (pH6.0, 0.1 mol / l) containing 5 mmol / l EDTA prepared immediately before use. The mixture is reacted at 37 ° C. for 1.5 hours. The reaction mixture is placed in a Sephadex G-25 column (1 x 30 cm) and eluted with sodium phosphate buffer (pH 6.0, 0.1 mol / l; containing 5 mol / l EDTA). The amount of Fab ′ obtained is 2.5 mg, calculated using an extinction coefficient of 1.48 g ⁻¹ L cm ⁻¹ and a molecular weight of 46,000 of F (ab ′) ₂ at 280 nm (J. Immunoassay, 4, 209-327 ( 1983).

[Preparation of peroxidase-labeled anti-α-hANP [17-28] Fab ′

2 mg horseradish peroxidase (50 mmol) is dissolved in 0.3 mol sodium phosphate buffer (pH 7.0, 0.1 mol / L). To this was added a mixture of 0.65 mg (2100 mmol) of N-succinimidyl 6-maleimidohexanoate and 0.03 ml of N, N-dimethylformamide, and the mixture was stirred at 30 ° C. for 0.5 to 1 hour to react. Let's do it. The reaction mixture is centrifuged to remove excess reagent by precipitation. The supernatant was passed through a Sephadex G-25 column (1.0 × 45 cm) and sodium phosphate buffer (pH 6.0, 0.1 mol / h) at a flow rate of 30-40 ml / hour so that the volume of each fraction was 0.5-1.0 ml. elute). Sephadex G-50 column (fine, Pharmacia) (1.0 × 6.4 cm, 5 mL) with fine mesh filter paper at the bottom is equilibrated with the buffer and centrifuged at 100 g × g for 2 min in test tube. The reaction product (0.5 mL) is placed in a column and centrifuged in the same manner. The obtained fractions are concentrated by centrifugation at 2,000 x g, 4 ° C with a microconcentrator (CENTRICON 30, manufactured by Amicon Corp.).

1.8 mg (45 mmol) of the maleimide peroxidase binder thus prepared is dissolved in sodium phosphate buffer (pH 6.0, 0.1 mol / L). To this was added about 2.0 mg (43 mmol) of Fab 'solution dissolved in 0.2-0.4 mL of sodium phosphate buffer (pH6.0, 0.1 mol / L) containing 5 mmol / L EDTA and the mixture at 20C. The reaction is carried out for 1 hour at 30 ° C. for 1 hour. The final concentration of maleimide peroxidase Fab 'conjugate in the reaction mixture is 50-100 mol / ml. The reaction mixture was passed through Ultrogel AcA 44 column (1.5 x 45 cm) and sodium phosphate buffer (pH6.5, 0.1 mL / min) at a flow rate of 0.3-0.5 mL / min so that the volume of each fraction was about 1.0 mL. elute). This yields about 2.5 mg of the desired peroxidase-labeled anti-α-hANP [17-28] Fab ′ (J. Immunoassay, 4, 209-327 (1983)).

[α-hANP [17-28] -nonspecific rabbit IgG conjugate]

105 mmol / L N- in a solution of α-hANP [17-28] (0.5 mg) dissolved in 0.2 mL sodium phosphate buffer (0.1 mol / L, pH7.0) in N, N′-dimethylformamide It is reacted with 0.01 ml of succinimidyl 6-maleimidohexanoate at 30 ° C. for 30 minutes. The reaction product is gel filtered through a Sephadex G-10 column (1.0 x 45 cm) using 0.1 mol / L sodium phosphate buffer (pH 6.0) containing 5 mmol / L EDTA. The average value of the maleimide group introduced into α-hANP [17-28] is 0.6 / molecule.

210 mmol / L S-acetyl mercaptosuccinic anhydride in which a non-specific rabbit IgG (10 mg) solution dissolved in 0.1 mol / L sodium phosphate buffer (0.6 mL, pH7.5) was dissolved in N, N′-dimethylformamide. 0.03 mL) and the solution is reacted at 30 ° C. for 30 minutes. 0.1 mol / l tris hydrochloride buffer (0.1 mol, pH 7.0), 0.1 mol / l EDTA (0.02 ml) and 1 mol / l hydroxylamine hydrochloride (0.1 ml, pH7.0) were added to the reaction mixture. The mixture is reacted at 30 ° C. for 5 minutes. The reaction product is gel filtered on a Sephadex G-25 column (1.0 x 45 cm) using 0.1 mol / l sodium phosphate buffer (pH 6.0) containing 5 mmol / l EDTA. The mean value of thiol groups introduced into nonspecific rabbit IgG is 11 / molecule.

A partial sample (2.0 ml) of the maleimide-α-hANP [17-28] was obtained from mercaptosuccinylation obtained in 0.1 mol / l sodium phosphate buffer (pH 6.0, 0.25 ml) containing 5 mmol / l EDTA. Incubate with nonspecific rabbit IgG (2.4 mg) at 30 ° C. for 30 minutes. 0.1 mL of N-ethylmaleimide 0.01 mmol / L is added to the reaction product to block the residual mercap group. The mixture is gel filtered onto a Sephadex G-25 column (1.0 x 45 cm) using 0.1 mol / L sodium phosphate buffer (pH 7.0). The average molecular number of α-hANP [17-28] bound to nonspecific rabbit IgG calculated from reduction of thiol group is 8.7 / molecule.

[Refining of Wasabi Peroxidase-labeled Anti-α-hANP [17-28] Fab ′]

α-hANP [17-28] -nonspecific rabbit IgG conjugate (2 mg), bovine tyroglobulin (10 mg) and mouse serum protein (20 mg) are combined with brominated cyanogen-activated Sepharose 4B (1 g), respectively. . Wasabi peroxidase-labeled anti-hANP [17-28] Fab ′ dissolved in 0.1 mol / l sodium phosphate buffer (pH6.5 0.8 ml) containing 1 g / l gelatin and 50 mg / l thimerosal. 7.8 mg) one bovine tyroglobulin-Sepharose 4B column at a flow rate of 0.5 mL / hour using 10 mmol / L sodium phosphate buffer (pH7.5) containing 1 g / L gelatin and 0.1 mol / L sodium chloride. (0.55 × 4.0 cm) and the other is passed through two columns of mouse serum protein-Sepharose 4B columns (0.55 × 4.0 cm). Then, further purification with α-hANP [17-28] -nonspecific rabbit IgG-Sepharose 4B column (0.35 × 2.0 cm) using 3.2 mmol / L hydrochloric acid (pH2.5). Eluent (1 mL) containing 0.35 mg of purified labeling material is immediately mixed with 0.1 mL of 1 mol / L sodium phosphate buffer (pH 7.0) and 0.01 mL of 100 g / L gelatin. The amount of labeling substance calculated in peroxidase activity is about 0.35 mg.

[Preparation of Monoclonal Anti-α-hANP-Coated Polystyrene Balls]

50 polystyrene balls (3.2 mm diameter, Precision Plastic Ball Co., Chicago, USA) were placed in 1 ml 0.1 M sodium phosphate buffer (pH 7.0) and 100 μg / ml monoclonal anti-α-hANP-IgG ₁ (KY-ANP-I) is added and the mixture is left overnight at room temperature. The polystyrene balls are washed with sodium phosphate buffer (pH 7.0) and then sodium azide is added at 0.1%. The polystyrene balls thus prepared are stored in a refrigerator.

[Collection of plasma]

Blood is collected in supine at 9:00 am from the main artery of a healthy male subject (26-32 years old) starved overnight. 40 mg furosemide is injected intravenously, allowed to walk for one hour, and blood is collected again from the same subject in the same manner. Blood is collected with a cold plastic syringe and transferred to a cold disposable siliconized glass tube with aprotinin and EDTA. The plasma is then separated by centrifugation (500 × g). Final concentrations of aprotinin and EDTA are 1,000 kallikrein deactivator units (KIU) / ml and 1 mg / ml, respectively.

[Enzyme-Immunoassay of α-hANP by Sandwich Method]

One monoclonal anti α-hANP IgG ₁ (KY-ANP-I) -coated polystyrene ball is placed in standard solution or plasma (total volume 0.15 mL) of α-hANP and left at 4 ° C. for 24 hours. α-hANP standard solution with 10 mM sodium phosphate buffer (pH7.0: 1 mg / ml gelatin, 0.3 M sodium chloride, 0.2 mM cystine, 1 mM EDTA, 1 mg / ml sodium azide, and 1000KIU / ml aprotinin) Dilute to a final volume of 0.15 ml. Plasma (50 μl) was added to 0.1 ml of 10 mM sodium phosphate buffer (pH 7.0: 1 mg / ml gelatin, 0.4 M sodium chloride, 0.3 mM cystine, 1.5 mM EDTA, 1.5 mg / ml sodium azide and 1,000 Kiu / ml afro). Containing tinine).

After removing the liquid fraction from the reaction product, the polystyrene balls are washed twice with 2 ml of 10 mM sodium phosphate buffer (pH7.0: 0.1 M sodium chloride). 50 mg of rabbit anti-α-hANP [17-28] Fab′-peroxidase conjugate purified by affinity chromatography and 0.15 mL of 10 mM sodium phosphate buffer (pH8.0: 1 mg / mL gelatin, 0.2 mM) Cystine and 1 mM EDTA), and the mixture is left at 4 ° C. for 24 hours. After removing the liquid fraction, the polystyrene ball is washed twice in the same manner as above and transferred to another test tube. In order to determine the activity of the conjugate peroxidase, after reacting with 3- (4-hydroxyphenyl) -propionic acid used as a substrate for 60 minutes at 30 ℃, the fluorescence intensity is measured. Fluorescence intensity is measured based on reference 200 mg / ml quinine / 50 mM sulfuric acid.

[Specificity]

In the EIA according to the present invention, the standard dilution curves of α-hANP are the same as the dilution curves of α-hANP [4-28], α-hANP [5-28] and α-hANP [7-28]. It also suggests no effect of N-terminal modification. On the other hand, deletion of the C-terminal amino acid significantly reduces reactivity. However, the terminal fragments: α-hANP [17-28] and α-hANP [1-6] show no response. Cross reactions with β-hANP and α-rANP are 4.7% and 0.01% on a molar basis, respectively. This result is consistent with the specificity of the antibody used: mouse monoclonal IgG ₁ immobilized on polystyrene balls is specific for the N-terminal half of the α-hANP ring structure, whereas rabbit Fab ′ bound to peroxidase specific for α-hANP [17-28].

[Effect of plasma]

Binding oxidase activity (nonspecific binding) measured without plasma and α-hANP was the same as peroxidase activation measured in the presence of plasma pretreated with ₁ mmol of monoclonal anti-α-hANP IgG ₁ (KY-ANP-I). Do. The recovery of α-hANP (10 to 200 pg / ml) applied to plasma containing 4.3 to 332 pg / ml ANP is 81 to 94%. The dilution curve of plasma is parallel to the standard curve of α-hANP obtained without plasma when the volume of plasma per test is in the range of 1-50 μl. Therefore, plasma interference is hardly observed when the volume of plasma is 50 μl or less. The results are shown in FIG.

[EIA sensitivity]

The detection limit of α-hANP is 30 fg (10 amol/tube). When 50 μl of plasma is used, the minimum detection limit of plasma α-hANP is 0.6 pg / ml (0.2 fmol / ml). the sensitivity of conventional RIA is much higher than the 10 to 10 degree ^twice.

[Reproducibility of EIA]

The reproducibility of the present invention is defined in five different steps over the range of 5-158 pg / ml dissolved in plasma. The number of abnormalities in reproducibility and intra-analysis reproducibility is 3.2 to 9.4% (n = 20) and 5.4 to 12.0%, respectively.

Plasma α-hANP levels in healthy subjects with basal and blood volume-constriction by EIA compared to that of RIA

The basal plasma α-hANP level determined by EIA for healthy subjects is 24.5 ± 5.9 pg / ml. After intravenous administration of furosemide (40 mg), the plasma α-hANP level in the blood volume-constricted state after 1 hour of walking decreased to 15.3 ± 3.7 pg / ml. Plasma α-hANP levels simultaneously determined by RIA were 28.2 ± 5.7 pg / ml and 18.3 ± 3.2 pg / ml, respectively. The following shows the relationship between the measurements of α-hANP by EIA (y) and RIA (x):

y = 0.78x + 1.3,

r = 0.92, n = 24

As can be clearly seen from the above analysis, the EIA by the method of the present invention is very sensitive and can measure plasma α-hANP concentration without extraction even in a blood volume-constricted state.

[Plasma α-hANP Levels in Cardiac Patients]

Plasma α-hANP levels in heart patients are measured in the α-hANP measuring reagent of the present invention and are shown in Table 1 below.

TABLE 1

Note: Patients 1 to 3 are serious heart patients.

[Application of One Step Method in EIA]

EIA in the present invention can also be performed in a one step method. 3 shows the standard and dilution curves of α-hANP following the one step method. In this method, hANP, enzyme-labeled antibody, and antibody-coated polystyrene ball are well mixed simultaneously and reacted at 4 ° C. for 24 hours. Peroxidase (POD) activity is measured at 30 ° C. for 30 minutes. The other conditions are identical to those of the two step method.

Claims (15)

A monoclonal antibody that recognizes the N-terminal half of the ring structure of α-hANP. The monoclonal antibody according to claim 1, which recognizes any part contained in α-hANP [7-16] and containing Met [12]. The monoclonal antibody according to claim 1, which is a monoclonal antibody KY-ANP-I produced by hybridoma KY-ANP-I (ECACC 87082001). The monoclonal antibody according to any one of claims 1 to 3, which is immobilized on a solid phase. The monoclonal antibody of claim 4, wherein the solid phase is free beads or polystyrene balls. A hybridoma producing the monoclonal antibody of any one of claims 1 to 3. The hybridoma of claim 6, wherein the hybridoma is KY-ANP-I (ECACC 87082001). One recognizes the N-terminal side of α-hANP and the other recognizes the C-terminal side of α-hANP, one containing two types of antibodies immobilized on a solid phase and the other labeled with an enzyme A reagent for immunoassay of α-hANP. The reagent for immunoassay according to claim 8, wherein the antibody is bound to the enzyme via a crosslinking agent. The reagent for immunoassay according to claim 8, wherein the enzyme is a peroxidase. The reagent for immunoassay according to claim 8, wherein the crosslinking agent is N-succinimidyl 4- (n-maleimidomethyl) cyclohexanoate or N-succinimidyl 6-maleimidohexanoate. The reagent for immunoassay according to claim 8, wherein the monoclonal antibody recognizing the N-terminal side of α-hANP is the antibody of any one of claims 1 to 3. The reagent for immunoassay according to claim 8, wherein the antibody recognizing the C-terminal side of α-hANP is an anti-α-hANP [17-28] antibody. The reagent for immunoassay according to claim 13, wherein the anti-α-hANP [17-28] antibody is obtained from rabbit anti-α-hANP [17-28] serum. The reagent for immunoassay according to claim 8, wherein the solid phase is free beads or polystyrene balls.

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